Figure 1.
Microfluidic flow assay and quantification of platelet accumulation.
A. Schematic of the microfluidic flow assay. Four channels (h = 50 µm, w = 500 µm) were placed over a patch of type 1 collagen. Blood was pipetted in an inlet well (large circle) and withdrawn through the outlet (small circle) at a constant flow rate to achieve the desired wall shear rate. Platelet accumulation was monitored at the upstream edge of the collagen patch by epifluorescence microscopy. B. Platelet surface coverage was measured over the course of 5 min at 150 s−1 (○), 300 s−1 (□), 750 s−1 (⋄) and 1500 s−1 (+). In addition to the final platelet surface coverage (SC), the lag time (LagT, black line) and the accumulation velocity (VPLT, red dotted line) were calculated from each curve. Lag time was defined as the time to 1% surface coverage. Accumulation velocity was defined as the slope of line SC versus time from tLAG to 5 min.
Table 1.
Characteristics of the cohort of donors.
Figure 2.
Sensitivity of platelet accumulation to collagen surface density.
Type 1 fibrillar collagen was adsorbed to clean glass slides at solution concentrations of 5–1000 µg/mL. Whole blood was perfused over the collagen substrates at 300 s−1 and platelet accumulation was measured by fractional surface coverage. There was significantly lower platelet accumulation on 5 µg/mL and 10 µg/mL surfaces than on surfaces prepared from solutions of greater than 50 µg/mL. Lines with ** denotes a p<0.01 for the Mann-Whitney U-test.
Table 2.
MFA intra-assay variation.
Figure 3.
Platelet accumulation as function of shear rate.
Platelets were labeled with a PE/Cy5 labeled mouse antihuman CD41a antibody and their accumulation was measured over the course of a 5 min flow assay. The top row shows the raw images and the bottom row shows the binary images following image processing at wall shear rates of 150 s−1 (A, A’), 300 s−1 (B, B’), 750 s−1 (C, C’) and 1500 s−1 (D, D’). Scale bar = 100 µm.
Figure 4.
Surface coverage, rate and lag time of platelet accumulation as a function of wall shear rate.
Recalcified citrated whole blood was perfused over type I collagen for 5 min and platelet accumulation was monitored as a function of wall shear rate and time (n = 50). Platelet accumulation was characterized by (A) percent surface coverage (SC) after 5 min, (B) the rate of platelet accumulation expressed as percent surface coverage per second (VPLT), and (C) the lag time to 1% surface coverage (LagT). Lines with ** denotes a p<0.01 for the Mann-Whitney U-test.
Figure 5.
Distribution of platelet surface coverage (SC) in cohort of normal donors.
Histogram of percent surface coverage (SC) for 150 s−1 (A), 300 s−1 (B), 750 s−1 (C), and 1500 s−1 (D) for the fifty normal donors.
Figure 6.
The effect of VWF plasma levels on platelet accumulation.
Platelet surface coverage increases with increasing VWF plasma levels at all wall shear rates. For each shear rate, bars represent the average platelet surface coverage in each quartile of VWF levels in a cohort of normal donors (n = 50).
Table 3.
Spearman correlation coefficient between VWF levels and platelet surface coverage (SC), lag time (LagT) and platelet accumulation velocity (VPLT).
Figure 7.
The effect of gender on platelet accumulation.
Differences in platelet surface coverage (SC) between men (black bars, n = 21) and women (white bars, n = 29) at each wall shear rate. Lines with ** denotes a p<0.01 for the Mann-Whitney U-test.
Figure 8.
The effect of GP6 genotype on platelet accumulation.
The AA genotype (white bars) yields higher accumulation than the AG genotype (black bars) at venous shear rates. Line with ** denotes a p<0.01, * denotes p<0.05 for the Mann-Whitney U-test.
Figure 9.
The effect of anticoagulant on platelet accumulation.
For each donor (n = 10), whole blood was collected into sodium citrate and CTI or CTI only. Line with ** denotes a p<0.01 for the Mann-Whitney U-test.
Figure 10.
The role of plasma protein adsorption on platelet accumulation.
Three conditions for each donor (n = 10) were considered; 5 min whole blood, 15 min whole blood, and 10 min plasma followed by 5 min whole blood. All conditions were performed at 1500 s−1. Line with ** denotes a p<0.01 for the Mann-Whitney U-test.1.